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In Vitro Effects of Taxol on Ciliogenesis in Quail Oviduct

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In Vitro Effects of Taxol on Ciliogenesis in Quail Oviduct In vitro effects of taxol on ciliogenesis in quail oviduct EMMANUELLE BOISVIEUX-ULRICH, MARIE-CHRISTINE LAINE and DANIEL SANDOZ Centre de Biologie Cellnlaire, CNRS, 67 rue Maurice Gunsbourg, 94200 Ivry-sur-Seine and University Pierre et Marie Curie, Paris, France Summary When induced by in vivo oestrogen stimulation, this area resulted from the binding of some vesicles ciliogenesis continues in culture in vitro of quail to the anchoring fibres of the basal body. They oviduct implants. Ultrastructure of ciliogenic cells fused in various numbers, occasionally forming a was compared after culture for 24 or 48 h in the ring, at the site of the transitional region, and presence or absence of 10~5 M-taxol. Taxol, which exhibited the characteristics of the ciliary necklace. promotes polymerization and stabilization of The association of basal bodies 'with vesicles or microtubules, disturbed ciliogenesis, but formation with the plasma membrane appeared to be a of basal bodies was unaffected by the drug. Con- necessary signal for in situ polymerization of axon- versely, their migration towards the apical surface emal doublets. seemed to be slowed down or blocked and axon- In addition, taxol induced polymerization of nu- emal doublets polymerized onto the distal end of merous microtubules in the cytoplasm, especially cytoplasmic basal bodies. They elongated and often in the apical part of the cell and in the Golgi area. constituted a more or less complete axoneme, This network of microtubules may prevent basal extending between organelles in various orien- body migration. tations. These axonemes, often abnormal, were not surrounded by a membrane, with the exception of the transitional or neck region between the basal body and axoneme. The formation of membrane in Key words: microtubules, taxol, ciliogenesis, quail oviduct. Introduction and B of basal bodies. Furthermore, microtubules associ- ated with the pericentriolar material are labile micro- Taxol, an experimental anti-tumour drug isolated from tubules. On the contrary, microtubules that polymerize Taxus brevifolia was shown to enhance microtubule onto basal bodies are stable doublets associated with assembly in vitro (Schiff et al. 1979) and in living cells numerous accessory proteins, all of them forming the (Schiff & Horwitz, 1980). The action of the drug is axoneme. specific to the microtubule system (Horwitz et al. 1982). Basal bodies of epithelial ciliated cells are mainly Cellular functions that are mediated by microtubules, generated near the Golgi apparatus (Sorokin, 1968; such as cell motility and cell shape, are modified by the Sandoz et al. 1976; Chang et al. 1979) and migrate action of taxol, which induces a marked change in towards the apical membrane, on which they bind microtubule distribution (Mazurovsky et al. 1982). through anchoring fibres (Anderson, 1972). While the It has been reported that taxol induced a loss of the mechanism of this polarized migration is not yet under- microtubule-organizing centre activity of centrosomes stood, myosin has been proposed as being involved in this (De Brabander et al. 1981). Because of the structural transport (Lemullois et al. 1987, 1988; Boisvieux-Ulrich analogy of basal bodies with centrioles, it was of interest etal. 1987). to compare their response to taxol treatment with that of In quail oviduct, ciliogenesis is easily induced by in centrioles. vivo oestrogen stimulation (Sandoz et al. 1975). Once Basal bodies differ from centrioles in their accessory induced, ciliogenesis continues in organotypic cultures in structures, i.e. anchoring fibres, basal foot and striated vitro (Boisvieux-Ulrich et al. 1987) and occurs as de- rootlet (Anderson & Brenner, 1971), and in the way in scribed under in vivo natural conditions (Chailley et al. which they induce tubulin polymerization. Microtubule 1982). Organotypic culture permits the study of modifi- polymerization is initiated from the pericentriolar ma- cations induced in ciliogenesis by drugs acting on the terial of centrosomes, which is considered as the main cytoskeleton, as already shown for benzodiazepines microtubule-organizing centre (MTOC) of the cell. Con- (Boisvieux-Ulrich et al. 1987). In this article we report versely, it is initiated on the distal end of microtubules A the effects of taxol on ciliogenesis and emphasize the Journal of Cell Science 92, 9-20 (1989) Printed in Great Bntain © The Company of Biologists Limited 1989 necessary interaction of basal bodies with membrane for by the development of microvilli, but also by the emerg- the induction of microtubule doublet polymerization. ence of short cilia. Ciliated cells with mature cilia were rare (about 5 %). The other cells (70 %) were undifferen- tiated and characterized by short microvilli and an Materials and methods occasional central primary cilium. During in vitm cul- ture, the ciliogenetic events continued, resulting in a Preparation of animals slight increase in the number of ciliogenic cells (about Eighteen immature ovariectomized quails (Coturnix coturnix 30%) and a large increase in ciliated cells (about 50%) japonica) were used in this study. They were prestimulated by after 48 h of culture (Fig. 2). The number of non-ciliated four daily intramuscular injections of 20 ng of oestradiol ben- cells was reduced and their apical surfaces were hidden by zoate (Benzogynestryl, Roussel, France), which initiated ciliary the numerous cilia of neighbouring ciliated cells. differentiation in epithelial cells of the oviduct (Sandoz et al. Taxol added to the culture medium inhibited ciliogen- 1975). esis. The percentage of non-ciliated, ciliating and ciliated cells did not change, during the 48 h of culture Preparation of samples (Fig. 3A-C). Many undifferentiated cells remained Oviduct was removed and cut into small segments, which were unchanged, and retained their primary cilium (Fig. 3A). maintained in vitro in organotypic culture for 2-48 h. One sample of each oviduct was immediately fixed to be used as a The only sign of differentiation was the formation of a control for the differentiation state before culture. Other small few microvilli on some cells, but without significant segments were opened longitudinally and cultured in sterile formation of new cilia. In the few ciliated cells, the ciliary Petriperm dishes (Hereaus). membrane of several cilia was fused (Fig. 3B,C) and some elongating polycilia exhibited a very rigid form. Methods of culture TEM examination of thin sections revealed that in Implants were maintained in culture medium 199, Hanks' salts, numerous cells basal body formation or early ciliogenesis Hepes buffer (Eurobio, Paris, France), supplemented with could be detected at the beginning of culture. These cells 10% quail serum, 0-1% insulin, 1% antibiotics/antimycotics were characterized by fibrous granules close to the Golgi (1000 units ml~' penicillin, lOOOOunitsml" streptomycin, apparatus in the supranuclear region and assembling 1 25 jig ml" fungizone) (Gibco, Cergy-Pontoise, France). basal bodies (Fig. 4). They were concomitantly enriched Taxol was kindly provided by Dr Potier (Institut de Chimie in microvilli compared to undifferentiated cells. des Substances Naturelles, CNRS, Gif-sur-Yvette, France). 2 Ciliogenetic processes normally occurred during the Stock solution of 10~ M-taxol was prepared in dimethylsulph- oxide (DMSO) and stored at —20°C. The final concentration 24 h of control culture and caused an increase in the used in the culture medium was 10~ M. number of cells with developing basal bodies or in Control samples were cultured in the absence of taxol, but ciliogenesis. After their assembly in the Golgi area, the with an equal concentration of DMSO (0-2%). Incubation was basal bodies separated and migrated towards the apical carried out in a 95 % O2 and 5% CO2 atmosphere, at 39°C. surface. As in normal development, they possessed Samples were removed at 2, 6, 24, 30 and 48 h after the typical accessory appendages, composed of a dense gran- beginning of culture. ule in the central core, anchoring fibres at the distal end and a lateral basal foot. Then, the basal bodies anchored Electtvn microscopy to the apical plasma membrane and numerous cilia began The samples were routinely fixed for 2 h with 3 % glutaralde- to elongate (Fig. 5). After 48 h of control culture, numer- hyde in 0-05M-sodium cacodylate buffer, pH7-4, postfixed for ous cells were ciliated. During these stages of develop- 1 h in 1 % OSO4 in the same buffer and processed for TEM and ment, the cortical cytoplasm contained only a few micro- SEM observation, as described (Boisvieux-Ulrich et al. 1987). tubules with random distribution. In order to visualize microtubules and cytoskeleton, some 5 samples were permeated for 2min with 0-15% Triton X-100 When 10~ M-taxol was added to the culture medium, and 10~s M-taxol in cacodylate buffer before being fixed with ciliogenesis was inhibited, but not basal body formation, glutaraldehyde. since the number of mature basal bodies in the apical cytoplasm of numerous cells increased (Figs 6, 7). A long primary cilium was still present in some other cells, but Results the formation of new cilia was rare (Fig. 6). After culture in the presence of taxol either for 2 h or Ciliary differentiation was obtained in culture in vitro of for 48 h, the cytoplasm was full of microtubules, which implants of quail oviduct after in vivo oestrogenic stimu- were particularly abundant in the supranuclear region of lation. Since ciliogenesis is not synchronous, various ciliogenic cells. The organization of microtubules consti- stages of differentiation were found at the beginning of tuting an intricate network around the groups of basal culture. SEM investigation of the surface samples al- bodies was more easily revealed in permeated cells lowed us to evaluate statistically the percentage of non- (Fig. 7). Microtubules were also abundant in the Golgi ciliated, ciliating and ciliated cells at different times and area, where they ran parallel to the Golgi saccules conditions of the experiments, as reported (Boisvieux- (Fig. 8). This area contained fewer fibrous granules Ulrich et al.
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